Modulation of eIF4E-BP2 expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of eIF4E-BP2. The compositions comprise oligonucleotides, targeted to nucleic acid encoding eIF4E-BP2. Methods of using these compounds for modulation of eIF4E-BP2 expression and for diagnosis and treatment of diseases and conditions associated with expression of eIF4E-BP2 are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Application Ser. No.60/538,752, filed on Jan. 22, 2004, the entire contents of which areherein incorporated by reference.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of eIF4E-BP2. In particular, this invention relates toantisense compounds, particularly oligonucleotide compounds, which, inpreferred embodiments, hybridize with nucleic acid molecules encodingeIF4E-BP2. Such compounds are shown herein to modulate the expression ofeIF4E-BP2.

BACKGROUND OF THE INVENTION

Eukaryotic gene expression must be regulated such that cells can rapidlyrespond to a wide range of different conditions. The process of mRNAtranslation is one step at which gene expression is highly regulated. Inresponse to hormones, growth factors, cytokines and nutrients, animalcells generally activate translation in preparation for theproliferative response. The rate of protein synthesis typicallydecreases under stressful conditions, such as oxidative or osmoticstress, DNA damage or nutrient withdrawal. Activation or suppression ofmRNA translation occurs within minutes and control over this process isthought to be exerted at the initiation phase of protein synthesis(Rosenwald et al., Oncogene, 1999, 18, 2507-2517; Strudwick and Borden,Differentiation, 2002, 70, 10-22).

Translation initiation necessitates the coordinated activities ofseveral eukaryotic initiation factors (eIFs), proteins which areclassically defined by their cytoplasmic location and ability toregulate the initiation phase of protein synthesis. One of thesefactors, eukaryotic initiation factor 4E (eIF4E), is present in limitingamounts relative to other initiation factors and is one component of theeIF4F initiation complex, which is also comprised of the scaffoldprotein eIF4G and the RNA helicase eIF4A. In the cytoplasm, eIF4Ecatalyzes the rate-limiting step of cap-dependent protein synthesis byspecifically binding to the 5′ terminal 7-methyl GpppX cap structurepresent on nearly all mature cellular mRNAs, which serves to deliver themRNAs to the eIF4F complex. Once bound, the eIF4F complex scans from the5′ to the 3′ end of the cap, permitting the RNA helicase activity ofeIF4A to resolve any secondary structure present in the 5′ untranslatedregion (UTR), thus revealing the translation initiation codon andfacilitating ribosome loading onto the mRNA (Graff and Zimmer, Clin.Exp. Metastasis, 2003, 20, 265-273; Strudwick and Borden,Differentiation, 2002, 70, 10-22).

eIF4E availability for incorporation into the eIF4E complex is regulatedthrough phosphorylation as well as through the binding of inhibitoryproteins. eIF4E is a phosphoprotein that is phosphorylated on serine 209by the mitogen-activated protein kinase-interacting kinase Mnk1, as wellas by protein kinase C (Flynn and Proud, J. Biol. Chem., 1995, 270,21684-21688; Wang et al., J. Biol. Chem., 1998, 273, 9373-9377;Waskiewicz et al., Embo J., 1997, 16, 1909-1920). The inhibitoryeIF4E-binding proteins 1 and 2 (eIF4E-BP1 and eIF4E-BP2) act aseffective inhibitors of cap-dependent translation by competing witheIF4G for binding to the dorsal surface of eIF4E (Pause et al., Nature,1994, 371, 762-767; Ptushkina et al., Embo J., 1999, 18, 4068-4075).When complexed with bp1, eIF4E is not a substrate for phosphorylation byprotein kinase C or Mnk1, indicating that dissociation of bp1 from eIF4Eis a prerequisite for eIF4E phosphorylation (Wang et al., J. Biol.Chem., 1998, 273, 9373-9377; Whalen et al., J Biol Chem, 1996, 271,11831-11837). Phosphorylation of eIF4E increases its affinity for mRNAcaps, thus elevating translation rates (Waskiewicz et al., Mol. CellBiol., 1999, 19, 1871-1880).

eIF4E-BP2 (also known as PHAS-II; 4EBP2; 4E-binding protein 2; EIF4EBP2)was cloned through use of the eIF4E protein in probing a cDNA expressionlibrary (Hu et al., Proc Natl Acad Sci USA, 1994, 91, 3730-3734; Pauseet al., Nature, 1994, 371, 762-767). eIF4E-BP2 is ubiquitously expressedin human tissues, including heart, brain, placenta, lung, liver, kidneyand spleen, as well as adipose tissue and skeletal muscle, the majorinsulin-responsive tissues (Hu et al., Proc Natl Acad Sci USA, 1994, 91,3730-3734; Tsukiyama-Kohara et al., Genomics, 1996, 38, 353-363). Thehuman gene maps to chromosome 10q21-q22 (Tsukiyama-Kohara et al.,Genomics, 1996, 38, 353-363). The mouse bp1 gene consists of threeexons, spans approximately 20 kb and maps to mouse chromosome 10(Tsukiyama-Kohara et al., Genomics, 1996, 38, 353-363). The expressionof eIF4E-BP2 does not appear to be altered in mice bearing a systemicdisruption of bp1 (Blackshear et al., J Biol Chem, 1997, 272,31510-31514).

Rather than preventing the binding of eIF4E to mRNA caps, eIF4E-BP2prohibits the binding of eIF4E to eIF4G, thereby preventing formation ofa complex that is necessary for efficient binding and proper positioningof the 40S ribosomal subunit on the target mRNA. When eIF4E-BP2 is boundto eIF4E, eIF4E does not serve as a substrate for phosphorylation byprotein kinase C, suggesting that dissociation of eIF4E-BP2 from eIF4Eis a prerequisite for phosphorylation of eIF4E (Whalen et al., J BiolChem, 1996, 271, 11831-11837). The region to which eIF4E binds is acommon motif shared by eIF4G and eIF4E-BP2, and point mutations in thisregion of eIF4E-BP2 abolish binding to eIF4E (Mader et al., Mol CellBiol, 1995, 15, 4990-4997). Two conserved motifs are present in theeIF4E-BP2: the RAIP motif, which is found in the NH2-terminal region ofEIF4E-BP2 and the TOS motif, which is formed by the last five aminoacids of eIF4E-BP2 (Schalm and Blenis, Curr Biol, 2002, 12, 632-639; Teeand Proud, Mol Cell Biol, 2002, 22, 1674-1683).

Like eIF4E-BP1, insulin stimulates the phosphorylation of eIF4E-BP2 incultured cells, which promotes the release of eIF4E-BP2 from eIF4E andallows for cap-dependent translation to proceed (Ferguson et al., J BiolChem, 2003, 278, 47459-47465). Mitogen-activated protein kinase, themajor insulin-stimulated kinase in rat adipocytes, can phosphorylaterecombinant eIF4E-BP2 in vitro. However, treatment of 3T3-L1 ratadipocytes with rapamycin attenuates the effects of insulin on thephosphorylation of eIF4E-BP2, indicating that elements of the mTORsignaling pathway mediate the actions of insulin on eIF4E-BP2 (Lin andLawrence, J Biol Chem, 1996, 271, 30199-30204). Additionally, serine-65of eIF4E-BP2 represents an ideal consensus site for phosphorylation bycyclic AMP-dependent protein kinase. In rat 3T3-L1 adipocytes, whereinsulin or epidermal growth factor markedly increased thephosphorylation of eIF4E-BP2, compounds that increase cyclic AMPdecrease the amount of radiolabeled phosphate incorporated intoeIF4E-BP2, and attenuate the effects of insulin on increasing thephosphorylation of eIF4E-BP2. Incubation of eIF4E-BP2 with the catalyticsubunit of cyclic AMP-dependent protein kinase results in the rapidphosphorylation of eIF4E-BP2. Together, these data suggest thatincreasing cyclic AMP may selectively increase eIF4E-BP2 phosphorylation(Lin and Lawrence, J Biol Chem, 1996, 271, 30199-30204).

Induction of cellular differentiation and reduction of cellularproliferation are concomitant with a reduction in translation rates, asis observed in conjunction with differential regulation of eIF4E-BPsduring human myeloid cell differentiation. When induced to differentiateinto monocytes/macrophages, cells from the HL-60 promyelocytic leukemiacell or U-937 monoblastic cell lines exhibit a decrease in thephosphorylation of bp1. In contrast, when HL-60 cells are stimulated todifferentiate into granulocytic cells, the amount of bp1 is decreased,whereas phosphorylation of bp1 is not affected. Conversely, eIF4E-BP2levels are markedly increased. These findings suggest that translationmachinery is differentially regulated during human myeloid celldifferentiation (Grolleau et al., J Immunol, 1999, 162, 3491-3497).

The disregulation of signaling networks that promote cell proliferationis often observed in association with cancer (Lawrence and Abraham,Trends Biochem Sci, 1997, 22, 345-349). Expression of excess eIF4E-BP2in cells transformed by eIF4E or v-src results in significant reversionof the transformed phenotype, demonstrating that eIF4E-BP2 can functionas an inhibitor of cell growth (Rousseau et al., Oncogene, 1996, 13,2415-2420).

The U.S. Pat No. 6,410,715 describes a purified human nucleic acidsequence encoding a cellular component that binds to eIF4E comprising acoding sequence for the protein eIF4E-BP2, and discloses a method forscreening a non-hormone agent potentially useful to treat a hormonedisorder (Sonenberg et al., 2000).

Currently, there are no known therapeutic agents that target eIF4E-BP2.Consequently, there remains a long felt need for agents capable ofeffectively inhibiting eIF4E-BP2. Antisense technology is an effectivemeans of reducing the expression of specific gene products and thereforeis uniquely useful in a number of therapeutic, diagnostic and researchapplications for the modulation of eIF4E-BP2 expression.

The present invention provides compositions and methods for inhibitingeIF4E-BP2 expression.

SUMMARY OF THE INVENTION

The present invention is directed to antisense compounds, especiallynucleic acid and nucleic acid-like oligomers, which are targeted to anucleic acid encoding eIF4E-BP2, and which modulate the expression ofeIF4E-BP2. Pharmaceutical and other compositions comprising thecompounds of the invention are also provided. Further provided aremethods of screening for modulators of eIF4E-BP2 and methods ofmodulating the expression of eIF4E-BP2 in cells, tissues or animalscomprising contacting said cells, tissues or animals with one or more ofthe compounds or compositions of the invention. Methods of treating ananimal, particularly a human, suspected of having or being prone to adisease or condition associated with expression of eIF4E-BP2, thereby insome instances delaying onset of said disease or condition, are also setforth herein. Such methods comprise administering a therapeutically orprophylactically effective amount of one or more of the compounds orcompositions of the invention to the person in need of treatment.

DETAILED DESCRIPTION OF THE INVENTION A. Overview of the Invention

The present invention employs antisense compounds, preferablyoligonucleotides and similar species for use in modulating the functionor effect of nucleic acid molecules encoding eIF4E-BP2. This isaccomplished by providing oligonucleotides which specifically hybridizewith one or more nucleic acid molecules encoding eIF4E-BP2. As usedherein, the terms “target nucleic acid” and “nucleic acid moleculeencoding eIF4E-BP2” have been used for convenience to encompass DNAencoding eIF4E-BP2, RNA (including pre-mRNA and mRNA or portionsthereof) transcribed from such DNA, and also cDNA derived from such RNA.The hybridization of a compound of this invention with its targetnucleic acid is generally referred to as “antisense”. Consequently, thepreferred mechanism believed to be included in the practice of somepreferred embodiments of the invention is referred to herein as“antisense inhibition.” Such antisense inhibition is typically basedupon hydrogen bonding-based hybridization of oligonucleotide strands orsegments such that at least one strand or segment is cleaved, degraded,or otherwise rendered inoperable. In this regard, it is presentlypreferred to target specific nucleic acid molecules and their functionsfor such antisense inhibition.

The functions of DNA to be interfered with can include replication andtranscription.

Replication and transcription, for example, can be from an endogenouscellular template, a vector, a plasmid construct or otherwise. Thefunctions of RNA to be interfered with can include functions such astranslocation of the RNA to a site of protein translation, translocationof the RNA to sites within the cell which are distant from the site ofRNA synthesis, translation of protein from the RNA, splicing of the RNAto yield one or more RNA species, and catalytic activity or complexformation involving the RNA which may be engaged in or facilitated bythe RNA. One preferred result of such interference with target nucleicacid function is modulation of the expression of eIF4E-BP2. In thecontext of the present invention, “modulation” and “modulation ofexpression” mean either an increase (stimulation) or a decrease(inhibition) in the amount or levels of a nucleic acid molecule encodingthe gene, e.g., DNA or RNA. Inhibition is often the preferred form ofmodulation of expression and mRNA is often a preferred target nucleicacid.

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In the present invention,the preferred mechanism of pairing involves hydrogen bonding, which maybe Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,between complementary nucleoside or nucleotide bases (nucleobases) ofthe strands of oligomeric compounds. For example, adenine and thymineare complementary nucleobases which pair through the formation ofhydrogen bonds. Hybridization can occur under varying circumstances.

An antisense compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a loss of activity, and there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

In the present invention the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which a compound ofthe invention will hybridize to its target sequence, but to a minimalnumber of other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances and in the context ofthis invention, “stringent conditions” under which oligomeric compoundshybridize to a target sequence are determined by the nature andcomposition of the oligomeric compounds and the assays in which they arebeing investigated.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleobases of an oligomeric compound. For example,if a nucleobase at a certain position of an oligonucleotide (anoligomeric compound), is capable of hydrogen bonding with a nucleobaseat a certain position of a target nucleic acid, said target nucleic acidbeing a DNA, RNA, or oligonucleotide molecule, then the position ofhydrogen bonding between the oligonucleotide and the target nucleic acidis considered to be a complementary position. The oligonucleotide andthe further DNA, RNA, or oligonucleotide molecule are complementary toeach other when a sufficient number of complementary positions in eachmolecule are occupied by nucleobases which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of precise pairing orcomplementarity over a sufficient number of nucleobases such that stableand specific binding occurs between the oligonucleotide and a targetnucleic acid.

It is understood in the art that the sequence of an antisense compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure orhairpin structure). It is preferred that the antisense compounds of thepresent invention comprise at least 70%, or at least 75%, or at least80%, or at least 85% sequence complementarity to a target region withinthe target nucleic acid, more preferably that they comprise at least 90%sequence complementarity and even more preferably comprise at least 95%or at least 99% sequence complementarity to the target region within thetarget nucleic acid sequence to which they are targeted. For example, anantisense compound in which 18 of 20 nucleobases of the antisensecompound are complementary to a target region, and would thereforespecifically hybridize, would represent 90 percent complementarity. Inthis example, the remaining noncomplementary nucleobases may beclustered or interspersed with complementary nucleobases and need not becontiguous to each other or to complementary nucleobases. As such, anantisense compound which is 18 nucleobases in length having 4 (four)noncomplementary nucleobases which are flanked by two regions ofcomplete complementarity with the target nucleic acid would have 77.8%overall complementarity with the target nucleic acid and would thus fallwithin the scope of the present invention. Percent complementarity of anantisense compound with a region of a target nucleic acid can bedetermined routinely using BLAST programs (basic local alignment searchtools) and PowerBLAST programs known in the art (Altschul et al., J.Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7,649-656).

Percent homology, sequence identity or complementarity, can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Insome preferred embodiments, homology, sequence identity orcomplementarity, between the oligomeric and target is between about 50%to about 60%. In some embodiments, homology, sequence identity orcomplementarity, is between about 60% to about 70%. In preferredembodiments, homology, sequence identity or complementarity, is betweenabout 70% and about 80%. In more preferred embodiments, homology,sequence identity or complementarity, is between about 80% and about90%. In some preferred embodiments, homology, sequence identity orcomplementarity, is about 90%, about 92%, about 94%, about 95%, about96%, about 97%, about 98%, about 99% or about 100%.

B. Compounds of the Invention

According to the present invention, antisense compounds includeantisense oligomeric compounds, antisense oligonucleotides, siRNAs,external guide sequence (EGS) oligonucleotides, alternate splicers,primers, probes, and other oligomeric compounds which hybridize to atleast a portion of the target nucleic acid. As such, these compounds maybe introduced in the form of single-stranded, double-stranded, circularor hairpin oligomeric compounds and may contain structural elements suchas internal or terminal bulges or loops. Once introduced to a system,the compounds of the invention may elicit the action of one or moreenzymes or structural proteins to effect modification of the targetnucleic acid.

One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense compounds which are“DNA-like” elicit RNAse H. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. Similar roleshave been postulated for other ribonucleases such as those in the RNaseIII and ribonuclease L family of enzymes.

While the preferred form of antisense compound is a single-strandedantisense oligonucleotide, in many species the introduction ofdouble-stranded structures, such as double-stranded RNA (dsRNA)molecules, has been shown to induce potent and specificantisense-mediated reduction of the function of a gene or its associatedgene products. This phenomenon occurs in both plants and animals and isbelieved to have an evolutionary connection to viral defense andtransposon silencing.

The first evidence that dsRNA could lead to gene silencing in animalscame in 1995 from work in the nematode, Caenorhabditis elegans (Guo andKempheus, Cell, 1995, 81, 611-620).

Montgomery et al. have shown that the primary interference effects ofdsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci.USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanismdefined in Caenorhabditis elegans resulting from exposure todouble-stranded RNA (dsRNA) has since been designated RNA interference(RNAi). This term has been generalized to mean antisense-mediated genesilencing involving the introduction of dsRNA leading to thesequence-specific reduction of endogenous targeted mRNA levels (Fire etal., Nature, 1998, 391, 806-811). Recently, it has been shown that itis, in fact, the single-stranded RNA oligomers of antisense polarity ofthe dsRNAs which are the potent inducers of RNAi (Tijsterman et al.,Science, 2002, 295, 694-697).

The antisense compounds of the present invention also include modifiedcompounds in which a different base is present at one or more of thenucleotide positions in the compound. For example, if the firstnucleotide is an adenosine, modified compounds may be produced whichcontain thymidine, guanosine or cytidine at this position. This may bedone at any of the positions of the antisense compound. These compoundsare then tested using the methods described herein to determine theirability to inhibit expression of eIF4E-BP2 mRNA.

In the context of this invention, the term “oligomeric compound” refersto a polymer or oligomer comprising a plurality of monomeric units. Inthe context of this invention, the term “oligonucleotide” refers to anoligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid(DNA) or mimetics, chimeras, analogs and homologs thereof. This termincludes oligonucleotides composed of naturally occurring nucleobases,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for a targetnucleic acid and increased stability in the presence of nucleases.

While oligonucleotides are a preferred form of the antisense compoundsof this invention, the present invention comprehends other families ofantisense compounds as well, including but not limited tooligonucleotide analogs and mimetics such as those described herein.

The antisense compounds in accordance with this invention preferablycomprise from about 8 to about 80 nucleobases (i.e. from about 8 toabout 80 linked nucleosides). One of ordinary skill in the art willappreciate that the invention embodies compounds of 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases inlength.

In one preferred embodiment, the antisense compounds of the inventionare 13 to 50 nucleobases in length. One having ordinary skill in the artwill appreciate that this embodies compounds of 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases inlength.

In another preferred embodiment, the antisense compounds of theinvention are 15 to 30 nucleobases in length. One having ordinary skillin the art will appreciate that this embodies compounds of 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases inlength.

Particularly preferred compounds are oligonucleotides from about 12 toabout 50 nucleobases, even more preferably those comprising from about15 to about 30 nucleobases.

Antisense compounds 13-80 nucleobases in length comprising a stretch ofat least thirteen (13) consecutive nucleobases selected from within theillustrative antisense compounds are considered to be suitable antisensecompounds as well.

While oligonucleotides are one form of antisense compound, the presentinvention comprehends other oligomeric antisense compounds, includingbut not limited to oligonucleotide mimetics such as are described below.The compounds in accordance with this invention can comprise from about8 to about 80 nucleobases. In another embodiment, the oligonucleotide isabout 10 to 50 nucleotides in length. In yet another embodiment, theoligonucleotide is 12 to 30 nucleotides in length. In yet anotherembodiment, the oligonucleotide is 12 to 24 nucleotides in length. Inyet another embodiment, the oligonucleotide is 19 to 23 nucleotides inlength. Some embodiments comprise at least an 8-nucleobase portion of asequence of an oligomeric compound which inhibits expression ofeIF4E-BP1. dsRNA or siRNA molecules directed to eIF4E-BP1, and their usein inhibiting eIF4E-BP1 mRNA expression, are also embodiments within thescope of the present invention.

The oligonucleotides of the present invention also include variants inwhich a different base is present at one or more of the nucleotidepositions in the oligonucleotide. For example, if the first nucleotideis an adenosine, variants may be produced which contain thymidine (oruridine if RNA), guanosine or cytidine at this position. This may bedone at any of the positions of the oligonucleotide. Thus, a 20-mer maycomprise 60 variations (20 positions×3 alternates at each position) inwhich the original nucleotide is substituted with any of the threealternate nucleotides.

These oligonucleotides are then tested using the methods describedherein to determine their ability to inhibit expression of eIF4E-BP1mRNA.

Exemplary preferred antisense compounds include oligonucleotidesequences that comprise at least the 13 consecutive nucleobases from the5′-terminus of one of the illustrative preferred antisense compounds(the remaining nucleobases being a consecutive stretch of the sameoligonucleotide beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the oligonucleotide contains about 13to about 80 nucleobases). Similarly preferred antisense compounds arerepresented by oligonucleotide sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same oligonucleotide beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the oligonucleotide contains about 8 to about 80 nucleobases). Itis also understood that preferred antisense compounds may be representedby oligonucleotide sequences that comprise at least 13 consecutivenucleobases from an internal portion of the sequence of an illustrativepreferred antisense compound, and may extend in either or bothdirections until the oligonucleotide contains about 13 to about 80nucleobases.

One having skill in the art armed with the preferred antisense compoundsillustrated herein will be able, without undue experimentation, toidentify further preferred antisense compounds.

C. Targets of the Invention

“Targeting” an antisense compound to a particular nucleic acid molecule,in the context of this invention, can be a multistep process. Theprocess usually begins with the identification of a target nucleic acidwhose function is to be modulated. This target nucleic acid may be, forexample, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes eIF4E-BP2.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes). It isalso known in the art that eukaryotic and prokaryotic genes may have twoor more alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAtranscribed from a gene encoding eIF4E-BP2, regardless of thesequence(s) of such codons. It is also known in the art that atranslation termination codon (or “stop codon”) of a gene may have oneof three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “Translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions which may betargeted effectively with the antisense compounds of the presentinvention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, apreferred region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

Other target regions include the 5′ untranslated region (5′ UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene), and the 3′ untranslated region(3′ UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA (or corresponding nucleotides on the gene). The 5′ cap site of anmRNA comprises an N7-methylated guanosine residue joined to the 5′-mostresidue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap regionof an mRNA is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap site. It is alsopreferred to target the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions, may also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also preferred target sites. mRNA transcripts produced via theprocess of splicing of two (or more) mRNAs from different gene sourcesare known as “fusion transcripts”. It is also known that introns can beeffectively targeted using antisense compounds targeted to, for example,DNA or pre-mRNA.

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through theuse of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites. Within thecontext of the invention, the types of variants described herein arealso preferred target nucleic acids.

The locations on the target nucleic acid to which the preferredantisense compounds hybridize are hereinbelow referred to as “preferredtarget segments.” As used herein the term “preferred target segment” isdefined as at least an 8-nucleobase portion of a target region to whichan active antisense compound is targeted. While not wishing to be boundby theory, it is presently believed that these target segments representportions of the target nucleic acid which are accessible forhybridization.

While the specific sequences of certain preferred target segments areset forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional preferred target segments may beidentified by one having ordinary skill.

Target segments 8-80 nucleobases in length comprising a stretch of atleast eight (8) consecutive nucleobases selected from within theillustrative preferred target segments are considered to be suitable fortargeting as well.

Target segments can include DNA or RNA sequences that comprise at leastthe 8 consecutive nucleobases from the 5′-terminus of one of theillustrative preferred target segments (the remaining nucleobases beinga consecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases). Similarlypreferred target segments are represented by DNA or RNA sequences thatcomprise at least the 8 consecutive nucleobases from the 3′-terminus ofone of the illustrative preferred target segments (the remainingnucleobases being a consecutive stretch of the sane DNA or RNA beginningimmediately downstream of the 3′-terminus of the target segment andcontinuing until the DNA or RNA contains about 8 to about 80nucleobases). It is also understood that preferred antisense targetsegments may be represented by DNA or RNA sequences that comprise atleast 8 consecutive nucleobases from an internal portion of the sequenceof an illustrative preferred target segment, and may extend in either orboth directions until the oligonucleotide contains about 8 to about 80nucleobases. One having skill in the art armed with the preferred targetsegments illustrated herein will be able, without undue experimentation,to identify further preferred target segments.

Once one or more target regions, segments or sites have been identified,antisense compounds are chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

The oligomeric antisense compounds may also be targeted to regions ofthe target nucleobase sequence (e.g., such as those disclosed in Example13) comprising nucleobases 1-80, 81-160, 161-240, 241-320, 321-400,401-480, 481-560, 561-640, 641-720, 721-800, 801-880, 881-960, 961-1040,1041-1120, 1121-1200, 1201-1280, 1281-1360, 1361-1440, 1441-1520,1521-1600, 1601-1680, 1681-1760, 1761-1840, 1841-1920, 1921-2000,2001-2080, 2081-2160, 2161-2240, 2241-2320, 2321-2400, 2401-2480,2481-2560, 2561-2640, 2641-2720, 2721-2782, or any combination thereof.

In one embodiment of the invention, the antisense compounds are targetedto a nucleic acid molecule encoding human eIF4E-BP2, for examplenucleotides 146-165 in the 5′ UTR, nucleotides 372-391, 420-520 or544-593 in the coding region, nucleotides 589-608 in the stop codonregion, nucleotides 623-766, 803-940, 1105-1599, 1868-1887, 1900-1919,1962-1981, 2218-2242, 2377-2401, 2449-2490, 2536-2555 or 2578-2597 inthe 3′ UTR, all of SEQ ID NO: 4; nucleotides 8892-8911 and 11559-11937in intron 1, and nucleotides 17941-17960 in the intron 1:exon 2junction, all of SEQ ID NO: 25; nucleotides 2088-2107 in the 3′ UTR ofSEQ ID NO: 26; and nucleotides 697-716 in the 3′ UTR of SEQ ID NO: 27,wherein said compound inhibits the expression of human eIF4E-BP2 mRNA.

In another embodiment of the invention, the antisense compounds aretargeted to a nucleic acid molecule encoding mouse eIF4E-BP2, forexample nucleotides 9-105 in the 5′UTR; nucleotides 132-480 in thecoding region; nucleotides 473-492 in the stop codon region; andnucleotides 500-1175, 1222-1638, 1662-1780 in the 3′ UTR, all of SEQ IDNO: 11; nucleotides 365-384 in the 3′ UTR of SEQ ID NO: 107; andnucleotides 36-55 in the 5′ UTR of SEQ ID NO: 108; wherein said compoundinhibits the expression of mouse eIF4E-BP2 mRNA.

In a further embodiment of the invention, antisense compounds aretargeted to a nucleic acid molecule encoding rat eIF4E-BP2, for examplenucleotides 7-26 in the 5′UTR, nucleotides 7-151, 164-247, 270-313, or303-388 in the coding region; nucleotides 390-409 in the stop codonregion and nucleotides 402-490 in the 3′ UTR, all of SEQ ID NO: 18;

wherein said compound inhibits the expression of rat eIF4E-BP2 mRNA.

D. Screening and Target Validation

In a further embodiment, the “preferred target segments” identifiedherein may be employed in a screen for additional compounds thatmodulate the expression of eIF4E-BP2. “Modulators” are those compoundsthat decrease or increase the expression of a nucleic acid moleculeencoding eIF4E-BP2 and which comprise at least an 8-nucleobase portionwhich is complementary to a preferred target segment. The screeningmethod comprises the steps of contacting a preferred target segment of anucleic acid molecule encoding eIF4E-BP2 with one or more candidatemodulators, and selecting for one or more candidate modulators whichdecrease or increase the expression of a nucleic acid molecule encodingeIF4E-BP2. Once it is shown that the candidate modulator or modulatorsare capable of modulating (e.g. either decreasing or increasing) theexpression of a nucleic acid molecule encoding eIF4E-BP2, the modulatormay then be employed in further investigative studies of the function ofeIF4E-BP2, or for use as a research, diagnostic, or therapeutic agent inaccordance with the present invention.

The preferred target segments of the present invention may be also becombined with their respective complementary antisense compounds of thepresent invention to form stabilized double-stranded (duplexed)oligonucleotides.

Such double stranded oligonucleotide moieties have been shown in the artto modulate target expression and regulate translation as well as RNAprocessing via an antisense mechanism. Moreover, the double-strandedmoieties may be subject to chemical modifications (Fire et al., Nature,1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons etal., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282,430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95,15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir etal., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15,188-200). For example, such double-stranded moieties have been shown toinhibit the target by the classical hybridization of antisense strand ofthe duplex to the target, thereby triggering enzymatic degradation ofthe target (Tijsterman et al., Science, 2002, 295, 694-697).

The antisense compounds of the present invention can also be applied inthe areas of drug discovery and target validation. The present inventioncomprehends the use of the compounds and preferred target segmentsidentified herein in drug discovery efforts to elucidate relationshipsthat exist between eIF4E-BP2 and a disease state, phenotype, orcondition. These methods include detecting or modulating eIF4E-BP2comprising contacting a sample, tissue, cell, or organism with thecompounds of the present invention, measuring the nucleic acid orprotein level of eIF4E-BP2 and/or a related phenotypic or chemicalendpoint at some time after treatment, and optionally comparing themeasured value to a non-treated sample or sample treated with a furthercompound of the invention. These methods can also be performed inparallel or in combination with other experiments to determine thefunction of unknown genes for the process of target validation or todetermine the validity of a particular gene product as a target fortreatment or prevention of a particular disease, condition, orphenotype.

E. Kits, Research Reagents, Diagnostics, and Therapeutics

The antisense compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. Furthermore, antisense oligonucleotides, which are able to inhibitgene expression with exquisite specificity, are often used by those ofordinary skill to elucidate the function of particular genes or todistinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics, the compounds of the present invention,either alone or in combination with other compounds or therapeutics, canbe used as tools in differential and/or combinatorial analyses toelucidate expression patterns of a portion or the entire complement ofgenes expressed within cells and tissues.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more antisense compounds are compared to controlcells or tissues not treated with antisense compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compoundswhich affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression)(Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

The antisense compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodingeIF4E-BP2. For example, oligonucleotides that are shown to hybridizewith such efficiency and under such conditions as disclosed herein as tobe effective eIF4E-BP2 inhibitors will also be effective primers orprobes under conditions favoring gene amplification or detection,respectively. These primers and probes are useful in methods requiringthe specific detection of nucleic acid molecules encoding eIF4E-BP2 andin the amplification of said nucleic acid molecules for detection or foruse in further studies of eIF4E-BP2. Hybridization of the antisenseoligonucleotides, particularly the primers and probes, of the inventionwith a nucleic acid encoding eIF4E-BP2 can be detected by means known inthe art. Such means may include conjugation of an enzyme to theoligonucleotide, radiolabelling of the oligonucleotide or any othersuitable detection means. Kits using such detection means for detectingthe level of eIF4E-BP2 in a sample may also be prepared.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense compounds have beenemployed as therapeutic moieties in the treatment of disease states inanimals, including humans. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat antisense compounds can be useful therapeutic modalities that canbe configured to be useful in treatment regimes for the treatment ofcells, tissues and animals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder which can be treated by modulating the expression ofeIF4E-BP2 is treated by administering antisense compounds in accordancewith this invention. For example, in one non-limiting embodiment, themethods comprise the step of administering to the animal in need oftreatment, a therapeutically effective amount of a eIF4E-BP2 inhibitor.The eIF4E-BP2 inhibitors of the present invention effectively inhibitthe activity of the eIF4E-BP2 protein or inhibit the expression of theeIF4E-BP2 protein. In one embodiment, the activity or expression ofeIF4E-BP2 in an animal is inhibited by about 10%. Preferably, theactivity or expression of eIF4E-BP2 in an animal is inhibited by about30%. More preferably, the activity or expression of eIF4E-BP2 in ananimal is inhibited by 50% or more. Thus, the oligomeric antisensecompounds modulate expression of eIF4E-BP2 mRNA by at least 10%, by atleast 20%, by at least 25%, by at least 30%, by at least 40%, by atleast 50%, by at least 60%, by at least 70%, by at least 75%, by atleast 80%, by at least 85%, by at least 90%, by at least 95%, by atleast 98%, by at least 99%, or by 100%.

For example, the reduction of the expression of eIF4E-BP2 may bemeasured in serum, adipose tissue, liver or any other body fluid, tissueor organ of the animal. Preferably, the cells contained within saidfluids, tissues or organs being analyzed contain a nucleic acid moleculeencoding eIF4E-BP2 protein and/or the eIF4E-BP2 protein itself.

The antisense compounds of the invention can be utilized inpharmaceutical compositions by adding an effective amount of a compoundto a suitable pharmaceutically acceptable diluent or carrier. Use of thecompounds and methods of the invention may also be usefulprophylactically.

The compounds of the present inventions are inhibitors of eIF4E-BP2expression. Thus, the compounds of the present invention are believed tobe useful for treating metabolic diseases and conditions, particularlydiabetes, obesity, hyperlipidemia or metabolic syndrome X. The compoundsof the invention are also believed to be useful for preventing ordelaying the onset of metabolic diseases and conditions, particularlydiabetes, obesity, hyperlipidemia or metabolic syndrome X. Metabolicsyndrome, metabolic syndrome X or simply Syndrome X refers to a clusterof risk factors that include obesity, dyslipidemia, particularly highblood triglycerides, glucose intolerance, high blood sugar and highblood pressure. Scott, C. L., Am J Cardiol. 2003 Jul. 3; 92(1A):35i-42i.The compounds of the invention have surprisingly been found to beeffective for lowering blood glucose, including plasma glucose, and forlowering blood lipids, including serum lipids, particularly serumcholesterol and serum triglycerides. The compounds of the invention aretherefore particularly useful for the treatment, prevention and delay ofonset of type 2 diabetes, high blood glucose and hyperlipidemia.

F. Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base sometimesreferred to as a “nucleobase” or simply a “base”. The two most commonclasses of such heterocyclic bases are the purines and the pyrimidines.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Informing oligonucleotides, the phosphate groups covalently link adjacentnucleosides to one another to form a linear polymeric compound. In turn,the respective ends of this linear polymeric compound can be furtherjoined to form a circular compound, however, linear compounds aregenerally preferred. In addition, linear compounds may have internalnucleobase complementarity and may therefore fold in a manner as toproduce a fully or partially double-stranded compound. Withinoligonucleotides, the phosphate groups are commonly referred to asforming the internucleoside backbone of the oligonucleotide. The normallinkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

Modified Internucleoside Linkages (Backbones)

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones containing a phosphorusatom therein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriaminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more intemucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotideshaving inverted polarity comprise a single 3′ to 3′ linkage at the3′-most internucleotide linkage i.e. a single inverted nucleosideresidue which may be abasic (the nucleobase is missing or has a hydroxylgroup in place thereof). Various salts, mixed salts and free acid formsare also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos.: 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.:5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

Modified Sugar and Internucleoside Linkages-Mimetics

In other preferred antisense compounds, e.g., oligonucleotide mimetics,both the sugar and the internucleoside linkage (i.e. the backbone), ofthe nucleotide units are replaced with novel groups. The nucleobaseunits are maintained for hybridization with an appropriate targetnucleic acid. One such compound, an oligonucleotide mimetic that hasbeen shown to have excellent hybridization properties, is referred to asa peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified Sugars

Modified antisense compounds may also contain one or more substitutedsugar moieties. Preferred are antisense compounds, preferably antisenseoligonucleotides, comprising one of the following at the 2′ position:OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Otherpreferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Antisense compounds may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos.: 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

A further preferred modification of the sugar includes Locked NucleicAcids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety.The linkage is preferably a methylene (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

Natural and Modified Nucleobases

Antisense compounds may also include nucleobase (often referred to inthe art as heterocyclic base or simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). Modified nucleobasesinclude other synthetic and natural nucleobases such as 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynylderivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine,5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.and are presently preferred base substitutions, even more particularlywhen combined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205;5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588;6,005,096; and 5,681,941, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, which is commonly owned with theinstant application and also herein incorporated by reference.

Conjugates

Another modification of the antisense compounds of the inventioninvolves chemically linking to the antisense compound one or moremoieties or conjugates which enhance the activity, cellular distributionor cellular uptake of the oligonucleotide. These moieties or conjugatescan include conjugate groups covalently bound to functional groups suchas primary or secondary hydroxyl groups. Conjugate groups of theinvention include intercalators, reporter molecules, polyamines,polyamides, polyethylene glycols, polyethers, groups that enhance thepharmaco-dynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugate groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve uptake,enhance resistance to degradation, and/or strengthen sequence-specifichybridization with the target nucleic acid. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve uptake, distribution, metabolism or excretion of thecompounds of the present invention. Representative conjugate groups aredisclosed in International Patent Application PCT/US92/09196, filed Oct.23, 1992, and U.S. Pat. No. 6,287,860, the entire disclosure of whichare incorporated herein by reference. Conjugate moieties include but arenot limited to lipid moieties such as a cholesterol moiety, cholic acid,a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Antisense compounds of the invention may also be conjugated to activedrug substances, for example, aspirin, warfarin, phenylbutazone,ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen,carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,indomethicin, a barbiturate, a cephalosporin, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

Chimeric Compounds

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide.

The present invention also includes antisense compounds which arechimeric compounds. “Chimeric” antisense compounds or “chimeras,” in thecontext of this invention, are antisense compounds, particularlyoligonucleotides, which contain two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of an oligonucleotide compound. Chimeric antisense oligonucleotidesare thus a form of antisense compound. These oligonucleotides typicallycontain at least one region wherein the oligonucleotide is modified soas to confer upon the oligonucleotide increased resistance to nucleasedegradation, increased cellular uptake, increased stability and/orincreased binding affinity for the target nucleic acid. An additionalregion of the oligonucleotide may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as RNAseL which cleaves both cellularand viral RNA. Cleavage of the RNA target can be routinely detected bygel electrophoresis and, if necessary; associated nucleic acidhybridization techniques known in the art.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

G. Formulations

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos.: 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Foroligonucleotides, preferred examples of pharmaceutically acceptablesalts and their uses are further described in U.S. Pat. No. 6,287,860,which is incorporated herein in its entirety. For oligonucleotides,presently preferred examples of pharmaceutically acceptable saltsinclude but are not limited to (a) salts formed with cations such assodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine. Sodium salts are presently believed to be more preferred.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration. Pharmaceutical compositionsand formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Coated condoms, gloves and the like may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogenous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug which may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes which are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome comprises oneor more glycolipids or is derivatized with one or more hydrophilicpolymers, such as a polyethylene glycol (PEG) moiety. Liposomes andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Preferred formulations for topical administration include those in whichthe oligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA).

For topical or other administration, oligonucleotides of the inventionmay be encapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters, pharmaceutically acceptable salts thereof, and theiruses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety. Topical formulations are describedin detail in U.S. patent application Ser. No. 09/315,298 filed on May20, 1999, which is incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof Preferred bileacids/salts and fatty acids and their uses are further described in U.S.Pat. No. 6,287,860, which is incorporated herein in its entirety. Alsopreferred are combinations of penetration enhancers, for example, fattyacids/salts in combination with bile acids/salts. A particularlypreferred combination is the sodium salt of lauric acid, capric acid andUDCA. Further penetration enhancers include polyoxyethylene-9-laurylether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the inventionmay be delivered orally, in granular form including sprayed driedparticles, or complexed to form micro or nanoparticles. Oligonucleotidecomplexing agents and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. Oralformulations for oligonucleotides and their preparation are described indetail in U.S. application Ser. Nos. 09/108,673 (filed Jul. 1, 1998),Ser. No. 09/315,298 (filed May 20, 1999) and Ser. No. 10/071,822, filedFeb. 8, 2002, each of which is incorporated herein by reference in theirentirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more oligomeric compounds and one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited tocancer chemotherapeutic drugs such as daunorubicin, daunomycin,dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemo-therapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Alternatively, compositions ofthe invention may contain two or more antisense compounds targeted todifferent regions of the same nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

H. Dosing

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀s found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01 ugto 100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same. Each of the references, GenBank® accession numbers, andthe like recited in the present application is incorporated herein byreference in its entirety.

EXAMPLES Example 1

Design and Screening of Duplexed Antisense Compounds Targeting eIF4E-BP2

In accordance with the present invention, a series of nucleic acidduplexes comprising the antisense compounds of the present invention andtheir complements can be designed to target eIF4E-BP2. The nucleobasesequence of the antisense strand of the duplex comprises at least a8-nucleobase portion of an oligonucleotide in Table 1. The ends of thestrands may be modified by the addition of one or more natural ormodified nucleobases to form an overhang. The sense strand of the dsRNAis then designed and synthesized as the complement of the antisensestrand and may also contain modifications or additions to eitherterminus. For example, in one embodiment, both strands of the dsRNAduplex would be complementary over the central nucleobases, each havingoverhangs at one or both termini. Overhangs can range from 2 to 6nucleobases and these nucleobases may or may not be complementary to thetarget nucleic acid. In another embodiment, the duplexes may have anoverhang on only one terminus.

For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG and having a two-nucleobase overhang ofdeoxythymidine(dT) would have the following structure:

In another embodiment, a duplex comprising an antisense strand havingthe same sequence CGAGAGGCGGACGGGACCG may be prepared with blunt ends(no single stranded overhang) as shown:

The RNA duplex can be unimolecular or bimolecular; i.e., the two strandscan be part of a single molecule or may be separate molecules.

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands (or alternatively, thecomplementary portions of a single RNA strand in the case of aunimolecular duplex) are annealed. The single strands are aliquoted anddiluted to a concentration of 50 uM. Once diluted, 30 uL of each strandis combined with 15uL of a 5× solution of annealing buffer. The finalconcentration of said buffer is 100 mM potassium acetate, 30 mMHEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 uL.This solution is incubated for 1 minute at 90° C. and then centrifugedfor 15 seconds. The tube is allowed to sit for 1 hour at 37° C. at whichtime the dsRNA duplexes are used in experimentation. The finalconcentration of the dsRNA duplex is 20 uM. This solution can be storedfrozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the duplexed antisense compounds are evaluated for theirability to modulate eIF4E-BP2 expression.

When cells reached 80% confluency, they are treated with duplexedantisense compounds of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM™-1 reduced-serum medium(Invitrogen Life Technologies, Carlsbad, Calif.) and then treated with130 μL of OPTI-MEM™-1 containing 12 μg/mL LIPOFECTIN™ (Invitrogen LifeTechnologies, Carlsbad, Calif.) per 200 nM of the desired duplexantisense compound. After 5 hours of treatment, the medium is replacedwith fresh medium. Cells are harvested 16 hours after treatment, atwhich time RNA is isolated and target reduction measured by real-timePCR.

Example 2

Oligonucleotide Isolation

After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32+/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 3

Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a 96-well format. Phosphodiester intemucleotidelinkages were afforded by oxidation with aqueous iodine.Phosphorothioate intemucleotide linkages were generated by sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) inanhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 4

Oligonucleotide Analysis—96-Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 5

Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, ribonucleaseprotection assays, or real-time PCR.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.),penicillin 100 units per mL, and streptomycin 100 micrograms per mL(Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (e.g., Falcon-Primaria#353872, BD Biosciences, Bedford, Mass.) at a density of approximately7000 cells/well for use in oligonucleotide transfection experiments andreal-time PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Life Technologies,Carlsbad, Calif.) supplemented with 10% fetal bovine serum (InvitrogenLife Technologies, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Life Technologies,Carlsbad, Calif.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence. Cells were seeded onto96-well plates (e.g., Falcon-Primaria #353872, BD Biosciences, Bedford,Mass.) at a density of approximately 5000 cells per well for use inoligonucleotide transfection experiments and real-time PCR analysis.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville, Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville, Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

b.END Cells:

The mouse brain endothelial cell line b.END was obtained from Dr. WernerRisau at the Max Plank Instititute (Bad Nauheim, Germany). b.END cellswere routinely cultured in DMEM, high glucose (Invitrogen LifeTechnologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum(Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (e.g., Falcon-Primaria#3872, BD Biosciences, Bedford, Mass.) at a density of approximately3000 cells/well for use in oligonucleotide transfection experiments andreal-time PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A10 Cells:

The rat aortic smooth muscle cell line A10 was obtained from theAmerican Type Culture Collection (Manassas, Va.). A10 cells wereroutinely cultured in DMEM, high glucose (American Type CultureCollection, Manassas, Va.) supplemented with 10% fetal bovine serum(Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinelypassaged by trypsinization and dilution when they reached 80%confluence. Cells were seeded into 96-well plates (e.g., Falcon-Primaria#3872, BD Biosciences, Bedford, Mass.) at a density of approximately2500 cells/well for use in oligonucleotide transfection experiments andreal-time PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

EMT-6 Cells:

The mouse mammary epithelial carcinoma cell line EMT-6 was obtained fromAmerican Type Culture Collection (Manassus, Va.). They were grown inserial monolayer culture in DMEM, high glucose (Invitrogen LifeTechnologies, Carlsbad, Calif.) supplemented with 10% fetal bovineserum, (Invitrogen Life Technologies, Carlsbad, Calif.), 100 ug/mlpenicillin and 100 ug/ml streptomycin (Invitrogen Life Technologies,Carlsbad, Calif.) in a humidified atmosphere of 90% air-10% CO₂ at 37°C. Cells were routinely passaged by trypsinization and dilution whenthey reached 85-90% confluencey. Cells were seeded into 96-well plates(e.g., Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at adensity of approximately 1000 cells/well for use in oligonucleotidetransfection experiments and real-time PCR analysis.

Treatment with Antisense Compounds:

When cells reached 65-75% confluency, they were treated witholigonucleotide.

Oligonucleotide was mixed with LIPOFECTIN™ (Invitrogen LifeTechnologies, Carlsbad, Calif.) in OPTI-MEM™-1 reduced-serum medium(Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desiredconcentration of oligonucleotide and a concentration of 2.5 to 3 ug/mLLIPOFECTIN™ per 100 nM oligonucleotide. For cells grown in 96-wellplates, wells were washed once with 100 μL OPTI-MEM™-1 reduced-serummedium and then treated with 130 μL of the LIPOFECTIN™/oligonucleotidemixture. Cells are treated and data are obtained in duplicate ortriplicate. After 4-7 hours of treatment at 37° C., the medium wasreplaced with fresh medium. Cells were harvested 16-24 hours afteroligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-rafThe concentration of positive control oligonucleotide that results in80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) orc-raf (for ISIS 15770) mRNA is then utilized as the screeningconcentration for new oligonucleotides in subsequent experiments forthat cell line. If 80% inhibition is not achieved, the lowestconcentration of positive control oligonucleotide that results in 60%inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. If 60% inhibition is not achieved, that particular cellline is deemed as unsuitable for oligonucleotide transfectionexperiments. The concentrations of antisense oligonucleotides usedherein are from 50 nM to 300 nM.

Example 6

Analysis of Oligonucleotide Inhibition of eIF4E-BP2 Expression

Antisense modulation of eIF4E-BP2 expression can be assayed in a varietyof ways known in the art. For example, eIF4E-BP2 mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR. Real-time quantitative PCR ispresently preferred. RNA analysis can be performed on total cellular RNAor poly(A)+ mRNA. The preferred method of RNA analysis of the presentinvention is the use of total cellular RNA as described in otherexamples herein. Methods of RNA isolation are well known in the art.Northern blot analysis is also routine in the art. Real-timequantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7600, 7700, or 7900 Sequence DetectionSystem, available from PE-Applied Biosystems, Foster City, Calif. andused according to manufacturer's instructions.

Protein levels of eIF4E-BP2 can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed toeIF4E-BP2 can be identified and obtained from a variety of sources, suchas the MSRS catalog of antibodies (Aerie Corporation, Birmingham,Mich.), or can be prepared via conventional monoclonal or polyclonalantibody generation methods well known in the art.

Example 7

Design of Phenotypic Assays for the use of eIF4E-BP2 Inhibitors

Once eIF4E-BP2 inhibitors have been identified by the methods disclosedherein, the compounds are further investigated in one or more phenotypicassays, each having measurable endpoints predictive of efficacy in thetreatment of a particular disease state or condition. Phenotypic assays,kits and reagents for their use are well known to those skilled in theart and are herein used to investigate the role and/or association ofeIF4E-BP2 in health and disease. Representative phenotypic assays, whichcan be purchased from any one of several commercial vendors, includethose for determining cell viability, cytotoxicity, proliferation orcell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston,Mass.), protein-based assays including enzymatic assays (Panvera, LLC,Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene ResearchProducts, San Diego, Calif.), cell regulation, signal transduction,inflammation, oxidative processes and apoptosis (Assay Designs Inc., AnnArbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis,Mo.), angiogenesis assays, tube formation assays, cytokine and hormoneassays and metabolic assays (Chemicon International Inc., Temecula,Calif.; Amersham Biosciences, Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated witheIF4E-BP2 inhibitors identified from the in vitro studies as well ascontrol compounds at optimal concentrations which are determined by themethods described above. At the end of the treatment period, treated anduntreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Measurement of the expression of one or more of the genes of the cellafter treatment is also used as an indicator of the efficacy or potencyof the eIF4E-BP2 inhibitors. Hallmark genes, or those genes suspected tobe associated with a specific disease state, condition, or phenotype,are measured in both treated and untreated cells.

Example 8

RNA Isolation

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem.,1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation areroutine in the art. Briefly, for cells grown on 96-well plates, growthmedium was removed from the cells and each well was washed with 200 μLcold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 MNaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added toeach well, the plate was gently agitated and then incubated at roomtemperature for five minutes. 55 μL of lysate was transferred to Oligod(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates wereincubated for 60 minutes at room temperature, washed 3 times with 200 μLof wash buffer (10 mM Tris-HCI pH 7.6, 1 mM EDTA, 0.3 M NaCl). After thefinal wash, the plate was blotted on paper towels to remove excess washbuffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mMTris-HCl pH 7.6), preheated to 70° C., was added to each well, the platewas incubated on a 90° C. hot plate for 5 minutes, and the eluate wasthen transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia, Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 150 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 150 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 1 minute. 500 μL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and incubatedfor 15 minutes and the vacuum was again applied for 1 minute. Anadditional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE wasthen added to each well of the RNEASY 96™ plate and the vacuum appliedfor a period of 90 seconds. The Buffer RPE wash was then repeated andthe vacuum was applied for an additional 3 minutes. The plate was thenremoved from the QIAVAC™ manifold and blotted dry on paper towels. Theplate was then re-attached to the QIAVAC™ manifold fitted with acollection tube rack containing 1.2 mL collection tubes. RNA was theneluted by pipetting 140 μL of RNAse free water into each well,incubating 1 minute, and then applying the vacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 9 Real-time Quantitative PCR Analysis of eIF4E-BP2 mRNA Levels

Quantitation of eIF4E-BP2 mRNA levels was accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., FAM or JOE, obtained from either PE-AppliedBiosystems, Foster City, Calif., Operon Technologies Inc., Alameda,Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 3′ end of the probe. When the probeand dyes are intact, reporter dye emission is quenched by the proximityof the 3′ quencher dye. During amplification, annealing of the probe tothe target sequence creates a substrate that can be cleaved by the5′-exonuclease activity of Taq polymerase. During the extension phase ofthe PCR amplification cycle, cleavage of the probe by Taq polymerasereleases the reporter dye from the remainder of the probe (and hencefrom the quencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™Sequence Detection System. In each assay, a series of parallel reactionscontaining serial dilutions of mRNA from untreated control samplesgenerates a standard curve that is used to quantitate the percentinhibition after antisense oligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

Isolated RNA is subjected to a reverse transcriptase (RT) reaction, forthe purpose of generating complementary DNA (cDNA), which is thesubstrate for the real-time PCR. Reverse transcriptase and real-time PCRreagents were obtained from Invitrogen Life Technologies, (Carlsbad,Calif.). The RT reaction and real-time PCR were carried out by adding 20μL PCR cocktail (2.5× PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM eachof dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverseprimer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM®Taq, 5 Units MuLV reverse transcriptase, and 2.5× ROX dye) to 96-wellplates containing 30 μL total RNA solution (20-200 ng). The RT reactionwas carried out by incubation for 30 minutes at 48° C. Following a 10minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles ofa two-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C for 1.5 minutes (annealing/extension).

Gene target quantities obtained by real-time PCR are normalized usingeither the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real timereal-time PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen™RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.).Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J.,et al, (Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nmand emission at 530 nm.

Probes and primers to human eIF4E-BP2 were designed to hybridize to ahuman eIF4E-BP2 sequence, using published sequence information (GenBank®accession number NM_(—)004096.3, incorporated herein as SEQ ID NO: 4).For human eIF4E-BP2 the PCR primers were:

-   forward primer: CCTCTAGTTTTGGGTGTGCATGT (SEQ ID NO: 5)-   reverse primer: CCCATAGCAAGGCAGAATGG (SEQ ID NO: 6) and the PCR    probe was: FAM-TGGAGTTTGTAGTGGGTGGTTTGTAAAACTGG-TAMRA (SEQ ID NO: 7)    where FAM is the fluorescent dye and TAMRA is the quencher dye. For    human GAPDH the PCR primers were:-   forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO: 8)-   reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 9) and the PCR    probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10)    where JOE is the fluorescent reporter dye and TAMRA is the quencher    dye.

Probes and primers to mouse eIF4E-BP2 were designed to hybridize to amouse eIF4E-BP2 sequence, using published sequence information (GenBank®accession number NM_(—)010124.1, incorporated herein as SEQ ID NO: 11).For mouse eIF4E-BP2 the PCR primers were:

-   forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 12)-   reverse primer: CGGACAGACGGACGATGAG (SEQ ID NO: 13) and the PCR    probe was: FAM-CCTCCCAGGTCTCTCGCCCT-TAMRA (SEQ ID NO: 14) where FAM    is the fluorescent reporter dye and TAMRA is the quencher dye. For    mouse GAPDH the PCR primers were:-   forward primer: GGCAAATTCAACGGCACAGT(SEQ ID NO: 15)-   reverse primer: GGGTCTCGCTCCTGGAAGAT(SEQ ID NO: 16) and the PCR    probe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID    NO: 17) where JOE is the fluorescent reporter dye and TAMRA is the    quencher dye.

Probes and primers to rat eIF4E-BP2 were designed to hybridize to a rateIF4E-BP2 sequence, using published sequence information (GenBank®accession number XM_(—)215414.1, incorporated herein as SEQ ID NO: 18).For rat eIF4E-BP2 the PCR primers were:

-   forward primer: AGTGAACAACTTGAACAACCTGAACA (SEQ ID NO: 19)-   reverse primer: ACTGCAGCAGGGTCAGATGTC (SEQ ID NO: 20) and the PCR    probe was: FAM-TCACGACAGGAAGCACGCAGTTGG-TAMRA (SEQ ID NO: 21) where    FAM is the fluorescent reporter dye and TAMRA is the quencher dye.    For rat GAPDH the PCR primers were:-   forward primer: TGTTCTAGAGACAGCCGCATCTT(SEQ ID NO: 22)-   reverse primer: CACCGACCTTCACCATCTTGT(SEQ ID NO: 23) and the PCR    probe was: 5′ JOE-TTGTGCAGTGCCAGCCTCGTCTCA-TAMRA 3′ (SEQ ID NO: 24)    where JOE is the fluorescent reporter dye and TAMRA is the quencher    dye.

Example 10

Northern Blot Analysis of eIF4E-BP2 mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

To detect human eIF4E-BP2, a human eIF4E-BP2 specific probe was preparedby PCR using the forward primer CCTCTAGTMTGGGTGTGCATGT (SEQ ID NO: 5)and the reverse primer CCCATAGCAAGGCAGAATGG (SEQ ID NO: 6). To normalizefor variations in loading and transfer efficiency membranes werestripped and probed for human glyceraldehyde-3-phosphate dehydrogenase(GAPDH) RNA (Clontech, Palo Alto, Calif.).

To detect mouse eIF4E-BP2, a mouse eIF4E-BP2 specific probe was preparedby PCR using the forward primer AGAGCAGCACAGGCTAAGACAGT (SEQ ID NO: 12)and the reverse primer CGGACAGACGGACGATGAG (SEQ ID NO: 13). To normalizefor variations in loading and transfer efficiency membranes werestripped and probed for mouse glyceraldehyde-3-phosphate dehydrogenase(GAPDH) RNA (Clontech, Palo Alto, Calif.).

To detect rat eIF4E-BP2, a rat eIF4E-BP2 specific probe was prepared byPCR using the forward primer AGTGAACAACTTGAACAACCTGAACA (SEQ ID NO: 19)and the reverse primer ACTGCAGCAGGGTCAGATGTC (SEQ ID NO: 20). Tonormalize for variations in loading and transfer efficiency membraneswere stripped and probed for rat glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMIAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 11

Antisense Inhibition of Human eIF4E-BP2 Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

In accordance with the present invention, a series of antisensecompounds was designed to target different regions of the humaneIF4E-BP2 RNA, using published sequences (GenBank® accession numberNM_(—)004096.3, incorporated herein as SEQ ID NO: 4, nucleotides20714677 to 20740000 of the sequence with GenBank® accession numberNT_(—)008583.16, incorporated herein as SEQ ID NO: 25, GenBank®accession number AK057643.1, incorporated herein as SEQ ID NO: 26,GenBank® accession number AK001936.1, incorporated herein as SEQ ID NO:27, and GenBank® accession number BF686401.1, incorporated herein as SEQID NO: 28). The compounds are shown in Table 1. “Target site” indicatesthe first (5′-most) nucleotide number on the particular target sequenceto which the compound binds. All compounds in Table 1 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-O-methoxyethyl (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on humaneIF4E-BP2 mRNA levels by quantitative realtime PCR as described in otherexamples herein. Data are averages from two experiments in which A549cells were treated with 75 nM of the antisense oligonucleotides of thepresent invention. SEQ ID NO: 2 was used as the control oligonucleotidein this assay. If present, “N.D.” indicates “no data”.

TABLE 1 Inhibition of human eIF4E-BP2 mRNA levels by chimeric phosphoro-thioate oligonucleotides having 2′-MOE wings and a deozy gap SEQ TARGETTARGET % ID ISIS # REGION SEQ ID NO SITE SEQUENCE INHIB NO 232773 Coding4 420 gccatgggagaattgcgacg 68 29 232776 Coding 4 493tttggagtcttcaattaagg 31 30 232777 Coding 4 498 tctactttggagtcttcaat 2 31232828 3′UTR 4 1962 gtctgtagtcatcttaaaaa 52 32 322947 Coding 4 501acttctactttggagtcttc 60 33 347546 Intron 1 25 1836 tagaccgcaggagctgcgaa0 34 347547 Intron 1 25 8892 agtgattctcaaactgcaga 38 35 347548 Intron 125 11559 tcttctgatccatggccacc 52 36 347549 Intron 1 25 11918tcagcactatctgttgaaaa 39 37 347550 Intron 1: Exon 2 25 16139attcgagttcctggaaaaca 0 38 junction 347551 Exon 2: Intron 2 25 16324ttctcttaccaactgcatgt 0 39 junction 347552 Intron 2: exon 25 17941gcatcatcccctagttagga 27 40 junction 347553 5′UTR 4 146cctcaggcggacggaaaagc 39 41 347554 Coding 4 332 cgggcgtggtgcaatagtca 6342 347555 Coding 4 372 attcgagttcctcccggtgt 55 43 347556 Coding 4 392gaaactttctgtcataaatg 0 44 347557 Coding 4 397 caacagaaactttctgtcat 15 45347558 Coding 4 474 gtgccagggctagtgactcc 43 46 347559 Coding 4 526attgttcaagttgttcaaat 0 47 347560 Coding 4 544 tgcatgtttcctgtcgtgat 59 48347561 Coding 4 549 ccaactgcatgtttcctgtc 54 49 347562 Coding 4 558gcatcatccccaactgcatg 54 50 347563 Coding 4 574 gtccatctcgaactgagcat 4651 347564 Stop Codon 4 589 gcaggagagtcagatgtcca 47 52 347565 3′UTR 4 623aagtatcagtgttgctgctt 45 53 347566 3′UTR 4 635 tcaggtgcacacaagtatca 43 54347567 3′UTR 4 734 atcatttggcacccagagga 54 55 347568 3′UTR 4 747agctcatcttcccatcattt 38 56 347569 3′UTR 4 772 acagggagaagaaatggtca 13 57347570 3′UTR 4 803 taacctgtttaactgggaag 59 58 347571 3′UTR 4 829cagaaatacagcaagggcct 70 59 347572 3′UTR 4 851 ctctaagggctgcttagctc 71 60347573 3′UTR 4 868 agagttgaactgttttcctc 78 61 347574 3′UTR 4 921caaaattacagggtatgagg 63 62 347575 3′UTR 4 1085 aagaccccaagcccagactc 9 63347576 3′UTR 4 1105 atttccccctgctggtttta 62 64 347577 3′UTR 4 1130aagggaaagcagctctcttt 70 65 347578 3′UTR 4 1180 agagttgcacaagctgtgct 4066 347579 3′UTR 4 1217 agtggacctcaaaacagtgt 64 67 347580 3′UTR 4 1303tctgcacaaatgcactaagt 65 68 347581 3′UTR 4 1350 aaaactggttaccaagggct 3469 347582 3′UTR 4 1357 gaagagcaaaactggttacc 27 70 347583 3′UTR 4 1393ccagcaacgagatgcaagca 65 71 347584 3′UTR 4 1410 agtacaagaggactctgcca 5672 347585 3′UTR 4 1458 tggtatggacctgctctagg 51 73 347586 3′UTR 4 1472gtgcctctattacttggtat 48 74 347587 3′UTR 4 1533 ttcttaggcattatctgaca 7075 347588 3′UTR 4 1541 agcggtcattcttaggcatt 59 76 347589 3′UTR 4 1580acgactgagaccgggtactc 67 77 347590 3′UTR 4 1614 acaactaccacaatgctcac 0 78347591 3′UTR 4 1664 attctgaaaatcaacttcaa 0 79 347592 3′UTR 4 1724tcccagcagccaaacaaagc 0 80 347593 3′UTR 4 1868 atttgaaaaatggcctggta 47 81347594 3′UTR 4 1892 acacttcaggtatctttgat 6 82 347595 3′UTR 4 1900agataccaacacttcaggta 49 83 347596 3′UTR 4 1912 acagatattctcagatacca 0 84347597 3′UTR 4 2018 atgtttaattaaaaagttgc 0 85 347598 3′UTR 4 2028acactggaagatgtttaatt 17 86 347599 3′UTR 4 2173 cagttttacaaaccacccac 0 87347600 3′UTR 4 2218 aagaatgaggctttcttgaa 47 88 347601 3′UTR 4 2223cagaaaagaatgaggctttc 34 89 347602 3′UTR 4 2246 tgaatgcaaaagcgaaaggg 0 90347603 3′UTR 4 2301 tcccgggattattatgctgc 0 91 347604 3′UTR 4 2377gaaattcccaggacaccagt 63 92 347605 3′UTR 4 2382 aaccagaaattcccaggaca 4793 347606 3′UTR 4 2389 caaatccaaccagaaattcc 0 94 347607 3′UTR 4 2449ccaaatggcctgttactctc 26 95 347608 3′UTR 4 2471 aacaaacaggtttctttctt 4096 347609 3′UTR 4 2492 cttttcatagttcaaaagaa 19 97 347610 3′UTR 4 2536cagacatccttcctctcttt 33 98 347611 3′UTR 4 2564 ttgtggcagaaaacagaaca 0 99347612 3′UTR 4 2578 aactattcacatttttgtgg 62 100 347613 3′UTR 4 2632tggagatccagcttattcct 49 101 347614 3′UTR 26 1189 aagaatgaaaagcttcattc 0102 347615 3′UTR 26 1336 tttaaatccattcctcaccg 0 103 347616 3′UTR 26 2088ataactaatacaggtggaag 41 104 347617 3′UTR 27 697 ggtcatctgaaatctctaaa 45105 347618 3′UTR 28 464 gcctcccacccttagaaagg 2 106

As shown in Table 1, SEQ ID NOs 29, 30, 32, 33, 35, 36, 37, 40, 41, 42,43, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 81, 83, 88, 89, 92, 93,95, 96, 98, 100, 101, 104 and 105 demonstrated at least 25% inhibitionof human EIF4E-BP2 expression in this assay and are therefore preferred.The target regions to which these preferred sequences are complementaryare herein referred to as “preferred target segments” and are thereforepreferred for targeting by compounds of the present invention. Thesepreferred target segments are shown in Table 5. These sequences areshown to contain thymine (T) but one of skill in the art will appreciatethat thymine (T) is generally replaced by uracil (U) in RNA sequences.The sequences represent the reverse complement of the preferredantisense compounds disclosed herein. “Target site” indicates the first(5′-most) nucleotide number on the particular target nucleic acid towhich the oligonucleotide binds. Also shown in Table 5 is the species inwhich each of the preferred target segments was found.

SEQ ID NOs 29, 30, 31 and 32 are cross species oligonucleotides whichare also complementary to the mouse eIF4E-BP2 nucleic acid target. SEQID NOs 29 and 33 are cross species oligonucleotides which are alsocomplementary to rat eIF4E-BP2.

Example 12

Antisense Inhibition of Mouse eIF4E-BP2 Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

In accordance with the present invention, a second series of antisensecompounds was designed to target different regions of the mouseeIF4E-BP2 RNA, using published sequences (GenBank® accession numberNM_(—)010124.1, incorporated herein as SEQ ID NO: 11, GenBank® accessionnumber B1696127.1, incorporated herein as SEQ ID NO: 107, and GenBank®accession number BE332409.1, incorporated herein as SEQ ID NO: 108). Thecompounds are shown in Table 2. “Target site” indicates the first(5′-most) nucleotide number on the particular target nucleic acid towhich the compound binds. All compounds in Table 2 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-O-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on mouseeIF4E-BP2 mRNA levels by quantitative real-time PCR as described inother examples herein. Data, shown in Table 2, are averages from twoexperiments in which b.END cells were treated with 150 nM of theantisense oligonucleotides of the present invention. SEQ ID NO: 2 wasused as the control oligonucleotide in this assay. If present, “N.D.”indicates “no data”.

TABLE 2 Inhibition of mouse eIF4E-BP2 mRNA levels in b.END cells bychimeric phosphorothioate oligonucleotides having 2′-MOE wings and adeoxy gap TAR- GET SEQ TAR- % SEQ ID GET IN- ID ISIS # REGION NO SITESEQUENCE HIB NO 232759 5′UTR 11 9 tctcaactcgcctgctctcg 92 109 2327605′UTR 11 26 ggctcctcacgctcggctct 81 110 232761 5′UTR 11 86tcgaggctttgtgcagcagc 64 111 232762 Coding 11 132 gctggtggctaccaccggcc 49112 232763 Coding 11 137 gctgggctggtggctaccac 72 113 232764 Coding 11179 gtcgctgatagccacggtgc 77 114 232765 Coding 11 201agtcctgaggtagctgcgcg 79 115 232766 Coding 11 211 gtggtgcagtagtcctgagg 81116 232767 Coding 11 264 cataaatgattcgtgttcct 73 117 232768 Coding 11269 tcggtcataaatgattcgtg 86 118 232769 Coding 11 274aactttcggtcataaatgat 52 119 232770 Coding 11 281 caacagaaactttcggtcat 72120 232771 Coding 11 286 cggtccaacagaaactttcg 84 121 232772 Coding 11299 gggagaattgcgacggtcca 80 122 232773 Coding 11 304gccatgggagaattgcgacg 83 29 232774 Coding 11 309 tctgcgccatgggagaattg 66123 232775 Coding 11 354 caggactggtgactccaggg 87 124 232776 Coding 11377 tttggagtcttcaattaagg 24 30 232777 Coding 11 382 tctactttggagtcttcaat69 31 232778 Coding 11 388 ttcacttctactttggagtc 71 125 232779 Coding 11449 aaactgagcctcatccccaa 89 126 232780 Coding 11 454atctcaaactgagcctcatc 85 127 232781 Coding 11 461 gatgtccatctcaaactgag 73128 232782 Stop 11 473 tggcagtagtcagatgtcca 91 129 Codon 232783 3′UTR 11500 ggctgctccacgaggcctcc 90 130 232784 3′UTR 11 521 tgggccagtcaggtgcacac77 131 232785 3′UTR 11 540 ctgtacactgtgttcctact 87 132 232786 3′UTR 11607 atgtgatcagacagtgcaca 67 133 232787 3′UTR 11 614 cgggaagatgtgatcagaca59 134 232788 3′UTR 11 696 ttcttctgtggactgtcagc 44 135 232789 3′UTR 11787 gtgctgcttggagactgccc 54 136 232790 3′UTR 11 798 tacaagcagaggtgctgctt47 137 232791 3′UTR 11 827 ggcactaaacctccttcacc 87 138 232792 3′UTR 11835 acacaatgggcactaaacct 68 139 232793 3′UTR 11 845 gagcccaggaacacaatggg61 140 232794 3′UTR 11 900 aatgtcccccacatccagcg 88 141 232795 3′UTR 11909 ctgaggacaaatgtccccca 81 142 232796 3′UTR 11 927 caggactgtgctccagagct78 143 232797 3′UTR 11 934 ggaggtacaggactgtgctc 69 144 232798 3′UTR 11975 gaggctgctgtcacatgtcc 68 145 232799 3′UTR 11 998 aagccttcctcccagagaaa81 146 232800 3′UTR 11 1020 tatcacacccaagacaagac 70 147 232801 3′UTR 111030 gatgatgagctatcacaccc 83 148 232802 3′UTR 11 1093cccttcaggagggcttaaaa 70 149 232803 3′UTR 11 1127 cagacaggcaaagaccagct 85150 232804 3′UTR 11 1156 tgcctacgggatgcaggtag 71 151 232805 3′UTR 111204 cttctgctctaaaagcagac 1 152 232806 3′UTR 11 1222caggccaaggtgttggcact 57 153 232807 3′UTR 11 1250 gctgagagcaggctggactc 66154 232808 3′UTR 11 1263 tctcaggcagaccgctgaga 54 155 232809 3′UTR 111276 gcccctgatgtattctcagg 72 156 232810 3′UTR 11 1282tcagaggcccctgatgtatt 51 157 232811 3′UTR 11 1289 gtcctcttcagaggcccctg 89158 232812 3′UTR 11 1303 tgcacggcggctcagtcctc 69 159 232813 3′UTR 111308 ctggctgcacggcggctcag 71 160 232814 3′UTR 11 1327aaaaccatgacccccgaggc 92 161 232815 3′UTR 11 1340 tacacctggttttaaaacca 67162 232816 3′UTR 11 1355 acacccaacgtaaggtacac 86 163 232817 3′UTR 111361 tgcaggacacccaacgtaag 85 164 232818 3′UTR 11 1381aaactcaaggtatagtaacc 73 165 232819 3′UTR 11 1392 aagtcgactttaaactcaag 66166 232820 3′UTR 11 1399 taagaggaagtcgactttaa 75 167 232821 3′UTR 111455 ctgtgctgctctctcagcag 21 168 232822 3′UTR 11 1467cactgtcttagcctgtgctg 90 169 232823 3′UTR 11 1584 tggaaaatggcccggtggaa 82170 232824 3′UTR 11 1619 tactaacatgggaggcatct 84 171 232825 3′UTR 111646 tgataaggagagactgatat 28 172 232826 3′UTR 11 1662taaaaggtctctcctctgat 33 173 232827 3′UTR 11 1668 taaaaataaaaggtctctcc 24174 232828 3′UTR 11 1682 gtctgtagtcatcttaaaaa 93 32 232829 3′UTR 11 1699aacttatctaaaaataggtc 30 175 232830 3′UTR 11 1708 tgtactgaaaacttatctaa 74176 232831 3′UTR 11 1749 atactggaagatgttttgtt 70 177 232832 3′UTR 111761 ataaccttcccaatactgga 82 178 232833 3′UTR 107 365acagctcatggcaaggcaga 79 179 232834 3′UTR 107 437 aactgctcttctatgtgtgg 4180 232835 3′UTR 107 454 tcgctgatagtctcttgaac 0 181 232836 5′UTR 108 36ggctcttcacgctcggctct 73 182

As shown in Table 2, SEQ ID NOs 29, 31, 32, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 169, 170, 171,176, 177, 178, 179 and 182 demonstrated at least 44% inhibition of mouseeIF4E-BP2 expression in this experiment and are therefore preferred. Thetarget regions to which these preferred sequences are complementary areherein referred to as “preferred target segments” and are thereforepreferred for targeting by compounds of the present invention. Thesepreferred target segments are shown in Table 4. These sequences areshown to contain thymine (T) but one of skill in the art will appreciatethat thymine (T) is generally replaced by uracil (U) in RNA sequences.The sequences represent the reverse complement of the preferredantisense compounds disclosed herein. “Target site” indicates the firstnucleotide number on the particular target nucleic acid to which theoligonucleotide binds. Also shown in Table 5 is the species in whicheach of the preferred target segments was found.

In a further embodiment, antisense oligonucleotides targeting mouseeIF4E-BP2 were tested in EMT-6 cells. The compounds were analyzed fortheir effect on mouse eIF4E-BP2 mRNA levels by quantitative real-timePCR as described in other examples herein. Data, shown in Table 3, areaverages from two experiments in which EMT-6 cells were treated with 150nM of the antisense oligonucleotides of the present invention. SEQ IDNO: 2 was used as the control oligonucleotide in this assay. If present,“N.D.” indicates “no data”.

TABLE 3 Inhibition of mouse eIF4E-BP2 mRNA levels in EMT-6 cells bychimeric phosphorothioate oligonucleotides having 2′-MOE wings and adeoxy gap TAR- GET SEQ TAR- % SEQ ID GET IN- ID ISIS # REGION NO SITESEQUENCE HIB NO 232759 5′UTR 11 9 tctcaactcgcctgctctcg 95 109 2327605′UTR 11 26 ggctcctcacgctcggctct 93 110 232761 5′UTR 11 86tcgaggctttgtgcagcagc 96 111 232762 Coding 11 132 gctggtggctaccaccggcc 88112 232763 Coding 11 137 gctgggctggtggctaccac 94 113 232764 Coding 11179 gtcgctgatagccacggtgc 95 114 232765 Coding 11 201agtcctgaggtagctgcgcg 97 115 232766 Coding 11 211 gtggtgcagtagtcctgagg 93116 232767 Coding 11 264 cataaatgattcgtgttcct 92 117 232768 Coding 11269 tcggtcataaatgattcgtg 98 118 232769 Coding 11 274aactttcggtcataaatgat 80 119 232770 Coding 11 281 caacagaaactttcggtcat 84120 232771 Coding 11 286 cggtccaacagaaactttcg 97 121 232772 Coding 11299 gggagaattgcgacggtcca 95 122 232773 Coding 11 304gccatgggagaattgcgacg 96 29 232774 Coding 11 309 tctgcgccatgggagaattg 93123 232775 Coding 11 354 caggactggtgactccaggg 98 124 232776 Coding 11377 tttggagtcttcaattaagg 73 30 232777 Coding 11 382 tctactttggagtcttcaat85 31 232778 Coding 11 388 ttcacttctactttggagtc 93 125 232779 Coding 11449 aaactgagcctcatccccaa 93 126 232780 Coding 11 454atctcaaactgagcctcatc 92 127 232781 Coding 11 461 gatgtccatctcaaactgag 89128 232782 Stop 11 473 tggcagtagtcagatgtcca 95 129 Codon 232783 3′UTR 11500 ggctgctccacgaggcctcc 98 130 232784 3′UTR 11 521 tgggccagtcaggtgcacac95 131 232785 3′UTR 11 540 ctgtacactgtgttcctact 98 132 232786 3′UTR 11607 atgtgatcagacagtgcaca 89 133 232787 3′UTR 11 614 cgggaagatgtgatcagaca75 134 232788 3′UTR 11 696 ttcttctgtggactgtcagc 59 135 232789 3′UTR 11787 gtgctgcttggagactgccc 77 136 232790 3′UTR 11 798 tacaagcagaggtgctgctt87 137 232791 3′UTR 11 827 ggcactaaacctccttcacc 91 138 232792 3′UTR 11835 acacaatgggcactaaacct 87 139 232793 3′UTR 11 845 gagcccaggaacacaatggg89 140 232794 3′UTR 11 900 aatgtcccccacatccagcg 95 141 232795 3′UTR 11909 ctgaggacaaatgtccccca 92 142 232796 3′UTR 11 927 caggactgtgctccagagct95 143 232797 3′UTR 11 934 ggaggtacaggactgtgctc 91 144 232798 3′UTR 11975 gaggctgctgtcacatgtcc 95 145 232799 3′UTR 11 998 aagccttcctcccagagaaa83 146 232800 3′UTR 11 1020 tatcacacccaagacaagac 80 147 232801 3′UTR 111030 gatgatgagctatcacaccc 91 148 232802 3′UTR 11 1093cccttcaggagggcttaaaa 85 149 232803 3′UTR 11 1127 cagacaggcaaagaccagct 94150 232804 3′UTR 11 1156 tgcctacgggatgcaggtag 95 151 232805 3′UTR 111204 cttctgctctaaaagcagac 36 152 232806 3′UTR 11 1222caggccaaggtgttggcact 83 153 232807 3′UTR 11 1250 gctgagagcaggctggactc 82154 232808 3′UTR 11 1263 tctcaggcagaccgctgaga 74 155 232809 3′UTR 111276 gcccctgatgtattctcagg 93 156 232810 3′UTR 11 1282tcagaggcccctgatgtatt 86 157 232811 3′UTR 11 1289 gtcctcttcagaggcccctg 95158 232812 3′UTR 11 1303 tgcacggcggctcagtcctc 86 159 232813 3′UTR 111308 ctggctgcacggcggctcag 91 160 232814 3′UTR 11 1327aaaaccatgacccccgaggc 96 161 232815 3′UTR 11 1340 tacacctggttttaaaacca 93162 232816 3′UTR 11 1355 acacccaacgtaaggtacac 95 163 232817 3′UTR 111361 tgcaggacacccaacgtaag 97 164 232818 3′UTR 11 1381aaactcaaggtatagtaacc 89 165 232819 3′UTR 11 1392 aagtcgactttaaactcaag 96166 232820 3′UTR 11 1399 taagaggaagtcgactttaa 96 167 232821 3′UTR 111455 ctgtgctgctctctcagcag 79 168 232822 3′UTR 11 1467cactgtcttagcctgtgctg 96 169 232823 3′UTR 11 1584 tggaaaatggcccggtggaa 96170 232824 3′UTR 11 1619 tactaacatgggaggcatct 95 171 232825 3′UTR 111646 tgataaggagagactgatat 60 172 232826 3′UTR 11 1662taaaaggtctctcctctgat 67 173 232827 3′UTR 11 1668 taaaaataaaaggtctctcc 23174 232828 3′UTR 11 1682 gtctgtagtcatcttaaaaa 98 32 232829 3′UTR 11 1699aacttatctaaaaataggtc 69 175 232830 3′UTR 11 1708 tgtactgaaaacttatctaa 97176 232831 3′UTR 11 1749 atactggaagatgttttgtt 89 177 232832 3′UTR 111761 ataaccttcccaatactgga 95 178 232833 3′UTR 107 365acagctcatggcaaggcaga 96 179 232834 3′UTR 107 437 aactgctcttctatgtgtgg 40180 232835 3′UTR 107 454 tcgctgatagtctcttgaac 23 181 232836 5′UTR 108 36ggctcttcacgctcggctct 88 182

As shown in Table 3,SEQ ID NOs 29, 30, 31, 32, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,173, 175, 176, 177, 178, 179 and 182 demonstrated at least 67%inhibition of mouse eIF4E-BP2 expression in this assay and are thereforepreferred.

Example 13

Antisense Inhibition of Rat eIF4E-BP2 Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

In accordance with the present invention, a third series of antisensecompounds was designed to target different regions of the rat eIF4E-BP2RNA, using published sequences (GenBank® accession numberXM_(—)215414.1, incorporated herein as SEQ ID NO: 18). The compounds areshown in Table 4. “Target site” indicates the first (5′-most) nucleotidenumber on the particular target nucleic acid to which the compoundbinds. All compounds in Table 4 are chimeric oligonucleotides(“gapmers”) 20 nucleotides in length, composed of a central “gap” regionconsisting of ten 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by five-nucleotide “wings”. The wings arecomposed of 2′-O-methoxyethyl (2′-MOE)nucleotides. The internucleoside(backbone) linkages are phosphorothioate (P═S) throughout theoligonucleotide. All cytidine residues are 5-methylcytidines. Thecompounds were analyzed for their effect on rat eIF4E-BP2 mRNA levels byquantitative real-time PCR as described in other examples herein. Data,shown in Table 4, are averages from two experiments in which A10 cellswere treated with 50 nM of the antisense oligonucleotides of the presentinvention. SEQ ID NO: 2 was used as the control oligonucleotide in thisassay. If present, “N.D.” indicates “no data”.

TABLE 4 Inhibition of rat eIF4E-BP2 mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTAR- GET SEQ TAR- % SEQ ID GET IN- ID ISIS # REGION NO SITE SEQUENCE HIBNO 232773 Coding 11 304 gccatgggagaattgcgacg 90 29 322907 5′UTR 18 7ggctcgtggctttgtgcagc 48 183 322908 Coding 18 48 tggtgtccaccaccggccga 49184 322909 Coding 18 57 tggctgggctggtgtccacc 65 185 322910 Coding 18 59tctggctgggctggtgtcca 50 186 322911 Coding 18 71 gaatggcgcggctctggctg 65187 322912 Coding 18 93 ctaatagccacggtgcgtgt 65 188 322913 Coding 18 97gtcgctaatagccacggtgc 83 189 322914 Coding 18 102 gctgcgtcgctaatagccac 80190 322915 Coding 18 114 tgaggtagctgcgctgcgtc 62 191 322916 Coding 18116 cctgaggtagctgcgctgcg 68 192 322917 Coding 18 120tagtcctgaggtagctgcgc 77 193 322918 Coding 18 122 agtagtcctgaggtagctgc 75194 322919 Coding 18 125 tgcagtagtcctgaggtagc 80 195 322920 Coding 18127 ggtgcagtagtcctgaggta 85 196 322921 Coding 18 130cgtggtgcagtagtcctgag 78 197 322922 Coding 18 132 ggcgtggtgcagtagtcctg 74198 322923 Coding 18 159 ggtgttgtggagaacagcgt 35 199 322924 Coding 18164 ctcccggtgttgtggagaac 48 200 322925 Coding 18 168gttcctcccggtgttgtgga 78 201 322926 Coding 18 193 aaactttcggtcataaatga 53202 322927 Coding 18 195 agaaactttcggtcataaat 41 203 322928 Coding 18197 acagaaactttcggtcataa 65 204 322929 Coding 18 198aacagaaactttcggtcata 79 205 322930 Coding 18 201 tccaacagaaactttcggtc 83206 322931 Coding 18 203 ggtccaacagaaactttcgg 83 207 322932 Coding 18208 gcgacggtccaacagaaact 80 208 322933 Coding 18 210ttgcgacggtccaacagaaa 76 209 322934 Coding 18 213 gaattgcgacggtccaacag 78210 322935 Coding 18 215 gagaattgcgacggtccaac 75 211 322936 Coding 18218 tgggagaattgcgacggtcc 36 212 322937 Coding 18 223cgccatgggagaattgcgac 73 213 322938 Coding 18 225 tgcgccatgggagaattgcg 52214 322939 Coding 18 228 gtctgcgccatgggagaatt 67 215 322940 Coding 18250 attgggcagatggcaaggtg 33 216 322941 Coding 18 265ggtgactccagggatattgg 35 217 322942 Coding 18 270 ggactggtgactccagggat 74218 322943 Coding 18 275 cgccaggactggtgactcca 83 219 322944 Coding 18292 ggagtcttccattaaggcgc 64 220 322945 Coding 18 294ttggagtcttccattaaggc 66 221 322946 Coding 18 298 tactttggagtcttccatta 27222 322947 Coding 18 303 acttctactttggagtcttc 68 33 322948 Coding 18 304cacttctactttggagtctt 64 223 322949 Coding 18 308 tgttcacttctactttggag 87224 322950 Coding 18 313 caagttgttcacttctactt 80 225 322951 Coding 18316 gttcaagttgttcacttcta 82 226 322952 Coding 18 323tcaggttgttcaagttgttc 83 227 322953 Coding 18 326 tgttcaggttgttcaagttg 84228 322954 Coding 18 329 gattgttcaggttgttcaag 68 229 322955 Coding 18332 cgtgattgttcaggttgttc 95 230 322956 Coding 18 335tgtcgtgattgttcaggttg 95 231 322957 Coding 18 339 ttcctgtcgtgattgttcag 88232 322958 Coding 18 341 gcttcctgtcgtgattgttc 95 233 322959 Coding 18343 gtgcttcctgtcgtgattgt 92 234 322960 Coding 18 348actgcgtgcttcctgtcgtg 97 235 322961 Coding 18 350 caactgcgtgcttcctgtcg 91236 322962 Coding 18 353 ccccaactgcgtgcttcctg 85 237 322963 Coding 18355 atccccaactgcgtgcttcc 48 238 322964 Coding 18 358ctcatccccaactgcgtgct 83 239 322965 Coding 18 360 gcctcatccccaactgcgtg 90240 322966 Coding 18 362 gagcctcatccccaactgcg 94 241 322967 Coding 18364 ctgagcctcatccccaactg 89 242 322968 Coding 18 369tcaaactgagcctcatcccc 50 243 322969 Stop 18 390 cagcagggtcagatgtccat 81244 Codon 322970 3′UTR 18 402 ccttcgacactgcagcaggg 88 245 322971 3′UTR18 406 gccgccttcgacactgcagc 83 246 322972 3′UTR 18 428gtgcacacgggccgtgtcag 76 247 322973 3′UTR 18 436 ccagtcaggtgcacacgggc 84248 322974 3′UTR 18 439 ggtccagtcaggtgcacacg 86 249 322975 3′UTR 18 443tactggtccagtcaggtgca 80 250 322976 3′UTR 18 446 tcctactggtccagtcaggt 72251 322977 3′UTR 18 450 gtgttcctactggtccagtc 84 252 322978 3′UTR 18 454cacggtgttcctactggtcc 83 253 322979 3′UTR 18 458 tgtacacggtgttcctactg 76254 322980 3′UTR 18 462 tctctgtacacggtgttcct 89 255 322981 3′UTR 18 464cttctctgtacacggtgttc 90 256 322982 3′UTR 18 469 tggagcttctctgtacacgg 90257 322983 3′UTR 18 471 actggagcttctctgtacac 85 258

As shown in Table 4, SEQ ID NOs 183, 184, 185, 186, 187, 188, 189, 190,191, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 204, 205, 206,207, 208, 209, 210, 211, 213, 214, 215, 218, 219, 220, 221, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257 and 258 demonstrated at least 48% inhibition ofrat eIF4E-BP2 expression in this experiment and are therefore preferred.The target regions to which these preferred sequences are complementaryare herein referred to as “preferred target segments” and are thereforepreferred for targeting by compounds of the present invention. Thesesequences are shown to contain thymine (T) but one of skill in the artwill appreciate that thymine (T) is generally replaced by uracil (U) inRNA sequences. The sequences represent the reverse complement of thepreferred antisense compounds shown in tables above. “Target site”indicates the first (5′-most) nucleotide number on the particular targetnucleic acid to which the compound binds.

“Preferred target segments,” as described in Table 5 of U.S. PatentApplication No. 60/538,752, filed Jan. 22, 2004, which is hereinincorporated by reference in its entirety, have been found byexperimentation to be open to, and accessible for, hybridization withthe antisense compounds of the present invention, one of skill in theart will recognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these preferred targetsegments and consequently inhibit the expression of eIF4E-BP2.

According to the present invention, antisense compounds includeantisense oligomeric compounds, antisense oligonucleotides, siRNAs,external guide sequence (EGS) oligonucleotides, alternate splicers,primers, probes, and other short oligomeric compounds which hybridize toat least a portion of the target nucleic acid.

Example 14

Western Blot Analysis of eIF4E-BP2 Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to eIF4E-BP2 is used,with a radiolabeled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Example 15

Reduction of Blood Glucose Levels in ob/ob Mice by Antisense Inhibitionof eIF4E-BP2

Ob/ob mice have a mutation in the leptin gene which results in obesityand hyperglycemia. As such, these mice are a useful model for theinvestigation of obesity and diabetes and treatments designed to treatthese conditions. In accordance with the present invention, compoundstargeted to eIF4E-BP2 are tested in the ob/ob model of obesity anddiabetes.

Seven-week old male C57B1/6J-Lepr ob/ob mice (Jackson Laboratory, BarHarbor, Me.) are fed a diet with a fat content of 10-15% and aresubcutaneously injected with oligonucleotides at a dose of 25 mg/kg twotimes per week for 4 weeks. Saline-injected animals, leptin wildtypelittermates (i.e. lean littermates) and ob/ob mice fed a standard rodentdiet serve as controls. After the treatment period, mice are sacrificedand target levels are evaluated in liver, brown adipose tissue (BAT) andwhite adipose tissue (WAT). RNA isolation and target mRNA expressionlevel quantitation are performed as described by other examples herein.

To assess the physiological effects resulting from antisense inhibitionof target mRNA, the ob/ob mice that receive antisense oligonucleotidetreatment are further evaluated at the end of the treatment period forserum lipids, serum free fatty acids, serum cholesterol, livertriglycerides, fat tissue triglycerides and liver enzyme levels. Hepaticsteatosis, accumulation of lipids in the liver, is assessed by measuringthe liver triglyceride content. Hepatic steatosis is assessed by routinehistological analysis of frozen liver tissue sections stained with oilred O stain, which is commonly used to visualize lipid deposits, andcounterstained with hematoxylin and eosin, to visualize nuclei andcytoplasm, respectively.

The effects of target inhibition on glucose and insulin metabolism areevaluated in the ob/ob mice treated with antisense oligonucleotides.Plasma glucose is measured at the start of the antisense oligonucleotidetreatment and following two and four weeks of treatment. Both fed andfasted plasma glucose levels were measured. At start of study, thetreatment groups of mice are chosen to have an average fed plasmaglucose level of about 350 mg/dL. Plasma insulin is also measured at thebeginning of the treatment, and following 2 weeks and 4 weeks oftreatment. Glucose and insulin tolerance tests are also administered infed and fasted mice. Mice receive intraperitoneal injections of eitherglucose or insulin, and the blood glucose and insulin levels aremeasured before the insulin or glucose challenge and at 15, 20 or 30minute intervals for up to 3 hours.

In mice treated with ISIS 232828 (SEQ ID NO: 32), an antisense inhibitorof eIF4E-BP2, fed plasma glucose levels were approximately 355 mg/dL atweek 0, 295 mg/dL at week 2 and 210 mg/dL at week 4. In contrast, micetreated with saline alone had fed plasma glucose levels of approximately365 mg/dL at week 0, 425 mg/dL at week 2 and 410 mg/dL at week 4. Micetreated with a positive control oligonucleotide, ISIS 116847(CTGCTAGCCTCTGGATTTGA; SEQ ID NO: 447), targeted to PTEN, had fed plasmaglucose levels of approximately 360 mg/dL at week 0, 215 mg/dL at week 2and 180 mg/dL at week 4.

Fasted plasma glucose was measured at week 3 of antisense treatment.Plasma glucose was approximately 330 mg/dL in saline treated mice, 245mg/dL in mice treated with ISIS 232828 (inhibitor of eIF4E-BP2) and 195mg/dL in mice treated with the positive control oligonucleotide, ISIS116847.

At the end of the four week study, average liver weights wereapproximately 3.6 grams for saline treated mice, 3.2 grams for ISIS232828-treated mice and 4.1 grams for positive control (ISIS 116847)treated mice. White adipose tissue weights were approximately 3.9 gramsfor saline treated mice, 3.8 grams for ISIS 232828-treated mice and 3.7grams for positive control (ISIS 116847) treated mice.

At the end of the study, liver transaminases were found to be lower inmice treated with antisense to eIF4E-BP2 (ISIS 232828) than in micetreated with saline or the positive control oligonucleotide (ISIS116847). AST levels were approximately 330 IU/L for saline treated mice,110 IU/L for ISIS 232828-treated mice and 430 IU/L for ISIS116847-treated mice. ALT levels were approximately 435 IU/L for salinetreated mice, 140 IU/L for ISIS 232828-treated mice and 710 IU/L forISIS 116847-treated mice.

Serum lipids were also measured at the end of the study. Cholesterollevels were approximately 230 mg/dL for saline treated mice, 210 mg/dLfor ISIS 232828-treated mice and 260 mg/dL for ISIS 116847-treated mice.Triglycerides were approximately 135 mg/dL for saline treated mice, 80mg/dL for ISIS 232828-treated mice and 110 mg/dL for ISIS 116847-treatedmice.

eIF4E-BP2 mRNA levels in liver were measured at the end of study usingRiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene,Oreg.) as taught in previous examples above. eIF4E-BP2 mRNA levels werereduced by approximately 90% in mice treated with ISIS 232828, comparedto saline treatment. Target reduction in mice treated with ISIS 116847was approximately 30%.

1. A compound comprising a modified oligonucleotide consisting of 15 to80 linked nucleosides and having a nucleobase sequence which is at least80% complementary to nucleotides 803-940 of SEQ ID NO: 4, wherein saidcompound inhibits the expression of human eIF4E-BP2 mRNA.
 2. Thecompound of claim 1 which is 15 to 50 nucleobases in length.
 3. Thecompound of claim 1 which is 15 to 30 nucleobases in length.
 4. Thecompound of claim 1 wherein the modified oligonucleotide comprises anantisesnse oligonucleotide.
 5. The compound of claim 1 wherein themodified oligonucleotide comprises a DNA oligonucleotide.
 6. Thecompound of claim 1 wherein the modified oligonucleotide comprises anRNA oligonucleotide.
 7. The compound of claim 1 wherein the modifiedoligonucleotide comprises an double-stranded oligonucleotide.
 8. Thecompound of claim 1 wherein the modified oligonucleotide comprises achimeric oligonucleotide.
 9. The compound of claim 1 wherein at least aportion of said compound hybridizes with RNA to form anoligonucleotide-RNA duplex.
 10. The compound of claim 1 having at least90% complementarity with nucleotides 803-940 of SEQ ID NO:
 4. 11. Thecompound of claim 1 having at least 95% complementarity with nucleotides803-940 of SEQ ID NO:
 4. 12. The compound of claim 1 having at least 99%complementarity with nucleotides 803-940 of SEQ ID NO:
 4. 13. Thecompound of claim 1 having at least one modified internucleosidelinkage, modified sugar, or modified nucleobase.
 14. The compound ofclaim 13 wherein at least one modified sugar comprises a2′-O-methoxyethyl.
 15. The compound of claim 13 wherein at least oneinternucleoside linkage is a phosphorothioate internucleoside linkage.16. A compound of claim 1 wherein said nucleobase sequence comprises atleast a 13-nucleobase portion of SEQ ID NO: 60 or
 61. 17. A compound ofclaim 1 wherein said nucleobase sequence is selected from the groupconsisting of SEQ ID NOs 60 or
 61. 18. The compound of claim 1 whereinsaid nucleobase sequence consists of SEQ ID NO: 60 or
 61. 19. Thecompound of claim 1, wherein said nucleobase sequence is complementaryto nucleotides 851-870 or nucleotides 868-887, both of SEQ ID No:
 4. 20.The compound of claim 1, wherein said nucleobase sequence iscomplementary to nucleotides 868-887 of SEQ ID NO:
 4. 21. The compoundof claim 15, wherein the modified nucleobase is a 5-methylcytosine. 22.The compound of claim 1 consisting of a single-stranded oligonucleotide.23. The compound of claim 1, wherein the modified oligonucleotidecomprises: a gap segment consisting of linked deoxynucleosides; a 5′wing segment consisting of linked nucleosides; a 3′ wing segmentconsisting of linked nucleosides; wherein the gap segment is positionedbetween the 5′ wing segment and the 3′ wing segment and wherein each ofsaid nucleosides of each of said wing segments comprises a modifiedsugar.
 24. The compound of claim 23, wherein the modifiedoligonucleotide comprises: a gap segment consisting of ten linkeddeoxynucleosides; a 5′ wing segment consisting of five linkednucleosides; a 3′ wing segment consisting of five linked nucleosides;wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment, wherein each of said nucleosides of each of saidwing segments comprises a 2′-O-methoxyethyl sugar; and wherein eachinternucleoside linkage is a phosphorothioate linkage.
 25. The compoundof claim 23, wherein the modified oligonucleotide consists of 20 linkednucleosides.
 26. A composition comprising a modified oligonucleotideconsisting of 15 to 30 linked nucleosides and having a nucleobasesequence comprising at least 12 contiguous nucleobases of a nucleobasesequence selected from among the nucleobase sequences recited in SEQ IDNOs: 60 and 61 or a salt thereof and a pharmaceutically acceptablecarrier or diluent.
 27. The composition of claim 26, wherein themodified oliginucleotide is a single-stranded oligonucleotide.
 28. Thecomposition of claim 26, wherein the modified oligonucleotide consistsof 20 linked nucleosides.
 29. A composition comprising the compound ofclaim 26 or a salt thereof and a pharmaceutically acceptable carrier ordiluent.
 30. The compound of claim 13, wherein the modified sugar is abicyclic sugar.